The idiotypic determinants expressed on the cell surface immunoglobulin (Ig) of B-cell lymphomas can act as tumour-associated antigens (for review see George and Stevenson, 1989). As such they present an attractive target for therapy, notably for the administration of passive anti-idiotypic antibody to patients (Miller et al., 1982 New Engl. J. Med. 306, 517–522). Murine monoclonal antibodies (MAbs) raised against idiotypic determinants on B cell non-Hodgkin's Lymphomas (NHLs) have given limited benefit in human therapeutic trials. Partial and complete responses have been observed, but the murine MAbs tend to recruit human effector functions inefficiently and are themselves the target of a human anti-mouse antibody response. Also, outgrowth of surface Ig negative lymphoma cells has been observed following therapy (Levy et al., 1987 J. Immunol. Rev. 96, 43-; Bahler and Levy, 1992 PNAS 89, 6770–6774), although the complete loss of immunoglobulin expression is rare (Meeker, et al., 1985 New Engl. J. Med. 312, 1658–1665; Zelentz et al., 1990 Ann. Oncol. 2, 115–122). In light of these limitations. coupled with the cost and inconvenience of generating MAbs for individual patients, the approach has not been widely adopted. However, it is clear that anti-idiotypic antibodies do have therapeutic potential in lymphomas.
One alternative to passive anti-idiotypic serotherapy is active immunisation which aims to break tolerance and induce a strong anti-idiotypic antibody response in the patient. Since the response will be polyclonal, it is more difficult for the target B-cell to escape selection, and furthermore, the response will be present on a continuing basis, and so might be able to control residual disease. An additional advantage of this approach is that it also has the potential to stimulate T cell mediated immune responses against the lymphoma. Efforts to stimulate tumour immunity using modified tumour cell vaccines have met with limited success, but active immunisation with idiotypic Ig prior to tumour challenge has proved effective in suppressing model B-cell tumours (Stevenson and Gordon, 1983 J. Immunol. 130, 970–973; George et al., 1987 J. Immunol. 138, 628–634; Campbell et al., 1987 J. Immunol. 139, 2825–2833) in animals and to treat animals bearing incipient tumour (George et al., 1988 J. Immunol. 141, 2168–2174). Furthermore idiotypic immunisation with human Ig isolated from patients with lymphoma has been associated with sustained tumour regression (Kwak et al., 1992 New Engl. J. Med. 327, 1209–1215).
The problem is how best to present the antigen (the idiotypic antibody) to break tolerance and stimulate an effective anti-lymphoma immune response, and this remains a challenge. In addition, for lymphomas, which secrete little immunoglobulin, making the idiotype is a major problem. To make sufficient idiotypic antibody for immunisation heterohybridomas must be prepared by fusion with mouse cell lines and the antibody then purified (Carroll et al., 1985 J. Immunol. Methods 89, 61–67). The yield is frequently low and it must be subsequently confirmed that the fusion derives from the human B-cell tumour.
This latter problem has now been overcome: the use of recombinant DNA technology allows the VH and VL genes encoding the idiotypic determinant to be readily identified in patient biopsy material by PCR and sequencing (Hawkins et al, 1994 Blood 83:3279). These genes can be assembled as scFv for use as a DNA vaccine. This approach is based on the data coming from a range of infectious diseases where it is clear that DNA encoding sequences from pathogens can transfect cells directly and induce protective immune responses (Ulmer et al, 1993259:1745; Davis & Whalen 1995, In Molecular and Cell Biology of Human Gene Therapeutics. Ed. George Dickson. Chapman & Hall, p368).
In previous work, in a mouse model for lymphoma, the V-genes of the tumour idiotope were cloned and expressed as light and heavy chain fusion proteins in bacteria. The separate chains were then used as immunogens. However, the separate chains were denatured, and in any case were not co-expressed to provide the paratope of the antibody. Indeed the authors suggested that “future work on peptides with fixed configurations similar to epitopes present in the native protein may prove useful, as may the co-expression of both VH and VL genes in bacteria to produce a recombinant Fv protein” (Campbell et al., 1987, cited above). However, the authors did not teach how to isolate the V-genes of the idiotope. Nor did the authors teach how to combine the recombinant Fv fragments into a vaccine.
The present inventors have previously shown that nucleic acid constructs can be prepared which can be delivered into living cells in vivo and which can then induce an immune response to an idiotypic determinant present on a malignant B-cell. The construct encodes a fusion protein comprising the idiotypic determinant and tetanus toxoid fragment C (FrC). Indeed, the inventors have already specifically shown that genetic fusion of a lymphoma or a myeloma-associated antigen to the adjuvant sequence FrC of tetanus toxin induces a protective immunity in mice against challenge with lymphoma or myeloma when used as a DNA vaccine.
However, the human population is immune to FrC due to vaccination with Tetanus Toxoid. Therefore, when FrC-based vaccine is used in patients it will work in a setting of pre-existing immunity to FrC. So far the inventors have found that pre-existing antibody does not reduce the response to the DNA vaccine. However, it is possible that different immune pathways are activated in this situation.